Long-term treatment of familial male-limited precocious puberty (testotoxicosis) with cyproterone acetate or ketoconazole

Background Familial male-limited precocious puberty (FMPP) or testotoxicosis is a rare gonadotrophin-independent form of sexual precocity caused by constitutively activating mutations of the LH receptor. Several clinical therapeutic approaches have been reported for this disorder, but with a paucity of long-term outcome data. Objective To evaluate the long-term treatment of testotoxicosis with cyproterone acetate or ketoconazole. Design A multicentric retrospective clinical study. Patients Ten boys from eight unrelated Brazilian families who carried known LH-receptor activating mutations were treated with 70 mg/m(2) cyproterone acetate (n = 5) or 10 mg/kg ketoconazole (n = 5) for a mean period of 5 and 8 years, respectively. Measurements Chronological and bone ages, bone age/chronological age ratio, target height (TH) range, adult height, basal and GnRH-stimulated gonadotrophin levels and basal testosterone levels were assessed. Results Growth velocity decreased significantly during treatment with cyproterone acetate or ketoconazole when compared to pretreatment value in each group (P < 0.05). Bone age/chronological age ratio decreased significantly after cyproterone acetate or ketoconazole therapy. Basal testosterone levels were significantly lower in patients undergoing ketoconazole compared to cyproterone acetate treatment [0.6 +/- 0.3 nmol/l (42 +/- 21 ng/dl) vs. 5.6 +/- 4.0 nmol/l (392 +/- 280 ng/dl); P < 0.05], as expected. Secondary gonadotrophin-dependent precocious puberty occurred at a similar frequency (40%) in both groups. Five patients have attained adult height and two patients have already reached 90% of their adult height. Two of them achieved their TH range and one patient, for whom TH was not available, had an adult height of 0.3 SDS. Four boys (two in each group) did not attain their TH range. Conclusion Long-term treatment with cyproterone acetate or ketoconazole resulted in similar outcomes without important side-effects in boys with testotoxicosis. However, both therapies showed limited efficacy in attaining normal adult height.

POSTOPERATIVE ANALGESIA INDUCED BY INTRATHECAL NEOSTIGMINE OR BETHANECHOL IN RATS

P>Cholinergic agonists and acetylcholinesterase inhibitors, such as neostigmine, produce a muscarinic receptor-mediated antinociception in several animal species that depends on activation of spinal cholinergic neurons. However, neostigmine causes antinociception in sheep only in the early, and not late, postoperative period. In the present study, a model of postoperative pain was used to determine the antinociceptive effects of bethanechol (a muscarinic agonist) and neostigmine administered intrathecally 2, 24 or 48 h after a plantar incision in a rat hind paw. Changes in the threshold to punctate mechanical stimuli were evaluated using an automated electronic von Frey apparatus. Mechanical hyperalgesia was obtained following plantar incision, the effect being stronger during the immediate (2 h) than the late post-surgical period. Bethanechol (15-90 mu g/5 mu L) or neostigmine (1-3 mu g/5 mu L) reduced incision-induced mechanical hyperalgesia, the effects of both drugs being more intense during the immediate (2 h) than the late post-surgical period. The ED(50) for bethanechol injected at 2, 24 and 48 h was 5.6, 51.9 and 82.5 mu g/5 mu L, respectively. The corresponding ED(50) for neostigmine was 1.62, 3.02 and 3.8 mu g/5 mu L, respectively. The decline in the antinociceptive potency of neostigmine with postoperative time is interpreted as resulting from a reduction in pain-induced activation of acetylcholine-releasing descending pathways. However, the similar behaviour of bethanechol in the same model points to an additional mechanism involving intrinsic changes in spinal muscarinic receptors.

Involvement of hypothalamic paraventricular nucleus non-N-methyl-d-aspartate receptors in the pressor response to noradrenaline microinjected into the bed nucleus of the stria terminalis of unanesthetized rats

Microinjection of noradrenaline into the bed nucleus of the stria terminalis (BST) has been reported to cause a pressor response in unanesthetized rats, which was shown to be mediated by acute vasopressin release into the systemic circulation. In the present study we verified the involvement of magnocellular neurons of the hypothalamic paraventricular (PVN) or supraoptic (SON) nuclei and the local neurotransmitter involved in the pressor response to noradrenaline microinjection into the BST. The PVN pretreatment with the non-selective neurotransmission blocker CoCl(2) (1 nmol/100 nL) inhibited the noradrenaline-evoked pressor response. However, responses were not affected by SON treatment with CoCl(2). Further experiments were carried out to test if glutamatergic neurotransmission in the PVN mediates the pressor response evoked by noradrenaline microinjection into the BST. Pretreatment of the PVN with the selective N-methyl-d-aspartate (NMDA) receptor antagonist LY235959 (2 nmol/100 nL) did not affect the noradrenaline-evoked pressor response. However, PVN pretreatment with the selective non-NMDA receptor antagonist NBQX (2 nmol/100 nL) significantly reduced the pressor response to noradrenaline microinjection into the BST. In conclusion, our results suggest that pressor responses to noradrenaline microinjection into the BST are mediated by PVN magnocellular neurons without involvement of SON neurons. They also suggest that a glutamatergic neurotransmission through non-NMDA glutamate receptors in the PVN mediates the response.

Evidence of the presence of T helper type 17 cells in chronic lesions of human periodontal disease

Periodontal disease is a chronic inflammation of the attachment structures of the teeth, triggered by potentially hazardous microorganisms and the consequent immune-inflammatory responses. In humans, the T helper type 17 (Th17) lineage, characterized by interleukin-17 (IL-17) production, develops under transforming growth factor-beta (TGF-beta), IL-1 beta, and IL-6 signaling, while its pool is maintained by IL-23. Although this subset of cells has been implicated in various autoimmune, inflammatory, and bone-destructive conditions, the exact role of T lymphocytes in chronic periodontitis is still controversial. Therefore, in this study we investigated the presence of Th17 cells in human periodontal disease. Gingival and alveolar bone samples from healthy patients and patients with chronic periodontitis were collected and used for the subsequent assays. The messenger RNA expression for the cytokines IL-17, TGF-beta, IL-1 beta, IL-6, and IL-23 in gingiva or IL-17 and receptor activator for nuclear factor-kappa B ligand in alveolar bone was evaluated by real-time polymerase chain reaction. The production of IL-17, TGF-beta, IL-1 beta, IL-6, and IL-23 proteins was evaluated by immunohistochemistry and the presence of Th17 cells in the inflamed gingiva was confirmed by immunofluorescence confocal microscopy for CD4 and IL-17 colocalization. Our data demonstrated elevated levels of IL-17, TGF-beta, IL-1 beta, IL-6, and IL-23 messenger RNA and protein in diseased tissues as well as the presence of Th17 cells in gingiva from patients with periodontitis. Moreover, IL-17 and the bone resorption factor RANKL were abundantly expressed in the alveolar bone of diseased patients, in contrast to low detection in controls. These results provided strong evidence for the presence of Th17 cells in the sites of chronic inflammation in human periodontal disease.

Glucocorticoid resistance in dialysis patients may impair the kidney allograft outcome

This study examines in vitro steroid sensitivity in chronic renal failure ( CRF) patients and its influence on the allograft outcome. We determined the inhibitory effect of dexamethasone ( DEX) on concanavalin A ( Con-A)-stimulated peripheral blood mononuclear cell ( PBMC) proliferation, and glucocorticoid receptor` ( GR) number of binding sites ( B-max) and affinity ( K-d) in 28 CRF patients and 40 normal healthy controls. Based on K-d values > 95th percentile from controls, patients were divided into two groups: glucocorticoid resistant ( n = 11) and glucocorticoid sensitive ( n = 17). Patients were followed during 18 months post-transplantation observing acute rejection episodes ( ARE), chronic allograft nephropathy ( CAN), allograft failure and death. The DEX concentration that caused 50% inhibition of Con-A-stimulated PBMC proliferation ( IC50) was higher in CRF than in healthy controls ( 2.2 x 10(-5) +/- 1.0 x 10(-5) versus 8.3 x 10(-6) +/- 4.2 x 10(-6) mol/ L, P = 0.02). Values of Kd ( 12.4 +/- 1.8 versus 7.2 +/- 0.9 nM) and Bmax ( 7.7 +/- 1.1 versus 4.1 +/- 0.3 fmol/ mg protein) were higher in CRF patients ( P = 0.02 and P = 0.001, respectively). There were higher incidences of ARE ( P = 0.02) and CAN ( P = 0.002) in the glucocorticoid-resistant group. Univariate and multivariate logistic regression showed that Kd was an independent predictor of ARE ( OR 8.8, P= 0.03) aswell as of CAN ( OR 16.5, P= 0.01). In conclusion, we observed glucocorticoid resistance in a subgroup of CRF patients undergoing dialysis, which led to a higher morbidity due to ARE and CAN in an 18-month follow-up period.

5-HT2C receptor regulation of defensive responses in the rat dorsal periaqueductal gray

Activation of 5-HT2C receptors in limbic structures such as the amygdala and hippocampus increases anxiety. Indirect evidence obtained with non-selective 5-HT2C-interacting drugs suggests that the same may occur in the dPAG, a brainstem region consistently implicated in the genesis/regulation of panic attacks. In this study we used more selective agonists and antagonists to unveil the role played by dPAG 5-HT2C receptors in the regulation of anxiety- and panic-related defensive behaviors. Our results showed that intra-dPAG microinjection of the endogenous agonist 5-HT (20 nmol) or the 5-HT2C receptor agonists MK-212 (1 and 10 nmol) and RO-600175 (40 nmol) significantly increased inhibitory avoidance acquisition in rats tested in the elevated T-maze, suggesting an anxiogenic effect. 5-HT, but not the two 5-HT2C receptor agonists, inhibited escape performance. In the elevated T-maze, inhibitory avoidance and escape responses have been related to generalized anxiety and panic attacks, respectively. The behavioral effects caused by 5-HT and MK-212 were fully blocked by previous local microinjection of the 5-HT2C receptor antagonist SB-242084. Intra-dPAG injection of MK-212 also failed to affect escape expression in another test relating this behavior to panic, the electrical stimulation of the dPAG. Overall, the results indicate that 5-HT2C receptors in the dPAG are preferentially involved in the regulation of defensive behaviors related to anxiety, but not panic. This finding extends to the dPAG the prominent role that has been attributed to 5-HT2C receptors in anxiety generation. (C) 2010 Elsevier Ltd. All rights reserved.

Recognition by toll-like receptor 2 induces antigen-presenting cell activation and Th1 programming during infection by Neospora caninum

Neospora caninum is an apicomplexan parasite responsible for major economic losses due to abortions in cattle. Toll-like receptors (TLRs) sense specific microbial products and direct downstream signaling pathways in immune cells, linking innate, and adaptive immunity. Here, we analyze the role of TLR2 on innate and adaptive immune responses during N. caninum infection. Inflammatory peritoneal macrophages and bone marrow-derived dendritic cells exposed to N. caninum-soluble antigens presented an upregulated expression of TLR2. Increased receptor expression was correlated to TLR2/MyD88-dependent antigen-presenting cell maturation and pro-inflammatory cytokine production after stimulation by antigens. Impaired innate responses observed after infection of mice genetically deficient for TLR2((-/-)) was followed by downregulation of adaptive T helper 1 (Th1) immunity, represented by diminished parasite-specific CD4(+) and CD8(+) T-cell proliferation, IFN-gamma:interleukin (IL)-10 ratio, and IgG subclass synthesis. In parallel, TLR2(-/-) mice presented higher parasite burden than wild-type (WT) mice at acute and chronic stages of infection. These results show that initial recognition of N. caninum by TLR2 participates in the generation of effector immune responses against N. caninum and imply that the receptor may be a target for future prophylactic strategies against neosporosis. Immunology and Cell Biology (2010) 88, 825-833; doi:10.1038/icb.2010.52; published online 20 April 2010

Expression of fibroblast growth factor 10 and its receptor, fibroblast growth factor receptor 2B, in the bovine corpus luteum

There is evidence that several fibroblast growth factors (FGFs) are involved in growth and development of the corpus luteum (CL), but many FGFs have not been investigated in this tissue, including FGF10. The objective of this study was to determine if FGF10 and its receptor (FGFR2B) are expressed in the CL. Bovine CL were collected from an abattoir and classed as corpus hemorrhagica (stage 1), developing (stage 11), developed (stage 111), and regressed (stage IV) CL. Expression of FGF10 and FGFR2B mRNA was measured by reverse transcription-polymerase chain reaction (RT-PCR). Both genes were expressed in bovine CL, and FGF10 expression did not differ between stages of CL development. FGF10 protein was localized to large and small luteal cells by immunohistochemistry. FGFR2B expression was approximately threefold higher in regressed compared to developing and developed CL (P < 0.05). To determine if FGF10 and FGFR2B expression is regulated during functional luteolysis, cattle were injected with PGF2 alpha and CL collected at 0, 0.5, 2, 4, 12, 24, 48, and 64 hr thereafter (n = 5 CL/time point), and mRNA abundance was measured by real-time RT-PCR. FGF10 mRNA expression did not change during functional luteolysis, whereas FGFR2B mRNA abundance decreased significantly at 2, 4, and 12 hr after PGF2a, and returned to pretreatment levels for the period 24-64 hr post-PGF2 alpha. These data suggest a potential role for FGFR2B signaling during structural luteolysis in bovine CL.

Isolation and partial characterization of Brazilian samples of feline immunodeficiency virus

Feline immunodeficiency virus (FIV) causes a slow progressive degeneration of the immune system which eventually leads to a disease comparable to acquired immune deficiency syndrome (AIDS) in humans. FIV has extensive sequence variation, a typical feature of lentiviruses. Sequence analysis showed that diversity was not evenly distributed throughout the genome, but was greatest in the envelope gene, env. The virus enters host cells via a sequential interaction, initiated by the envelope glycoprotein (env) binding the primary receptor molecule CD134 and followed by a subsequent interaction with chemokine co-receptor CXCR4. The purpose of this study was to isolate and characterize isolates of FIV from an open shelter in Sao Paulo, Brazil. The separated PBMC from 11 positive cats were co-cultured with MYA-1 cells. Full-length viral env glycoprotein genes were amplified and determined. Chimeric feline x human CD134 receptors were used to investigate the receptor utilization of 17 clones from Brazilian isolates of Fly. Analyses of the sequence present of molecular clones showed that all clones grouped within subtype B. In contrast to the virulent primary isolate FIV-GL8, expression of the first cysteine-rich domain (CRD1) of feline CD134 in the context of human CD134 was sufficient for optimal receptor function for all Brazilian FIV isolates tested. (C) 2011 Elsevier B.V. All rights reserved.

Plasticity of Opioid Receptors in the Female Periaqueductal Gray: Multiparity-Induced Increase in the Activity of Genes Encoding for Mu and Kappa Receptors and a Post-Translational Decrease in Delta Receptor Expression

The periaqueductal gray (PAG) has been reported as a potential site for opioid regulation of behavioral selection. Opioid-mediated behavioral and physiological responses differ between nulliparous and multiparous females. This study addresses the effects of multiple reproductive experiences on mu-, kappa- and delta-opioid receptor (Oprm1, Oprk1, and Oprd1 respectively) gene activity and mu, kappa and delta protein expression (MOR, KOR and DOR respectively) in the PAG of the female rats. This was done by evaluating the opioid gene expression using real-time (RT-PCR) and quantification of each protein receptor by Western blot analysis. The RT-PCR results show that multiple reproductive experiences increase Oprm1 and Oprk1 gene expression. Western blot analysis revealed increased MOR and KOR while DOR protein was decreased in multiparous animals. Taken together, these data suggest that multiple reproductive experiences influence both gene activity and opioid receptor expression in the PAG. Post-translational mechanisms seem particularly relevant for DOR expression. Thus, opioid transmission in the PAG might be modulated by different mechanisms of multiparity-induced plasticity according to the opioid receptor type.

Effect of Recombinant Bovine Somatotropin on Plasma Concentrations of Insulin-like Growth Factor I, Insulin and Membrane Integrity of Bull Spermatozoa

Contents This study aimed to evaluate the effect of the exogenous recombinant bovine somatotropin (rbST) on plasma concentrations of insulin-like growth factor I (IGF-I), insulin and semen quality of bulls. Twenty bulls (Aberdeen Angus and Brangus) were divided by breed into two groups. Placebo group was injected with NaCl 0.9% (s.c.) and treatment group with rbST (s.c., 500 mg) at days 0 and 14 of the experiment. Immediately after semen collection, blood samples were taken on days 0, 14, 28, 42 and 56 of the experiment. Semen was also collected on day 70 of the experiment. Evaluation of sperm motility was performed at pre-freezing and post-thawing stage, whereas assessment of sperm membrane integrity was performed after freezing and thawing. Analysis of data revealed that the effect of treatment and treatment-by-collection day on plasma concentrations of IGF-I and insulin was not significant. However, mean plasma concentrations of IGF-I and insulin were affected (p < 0.0001) by days of blood sampling. Effect of treatment and treatment-by-collection day on motility of spermatozoa was similar (p > 0.05) at pre-freezing and post-thawing stage. Intactness of plasmalemma and tail membrane of spermatozoa at post-thawing stage was higher (p < 0.05) in rbST-treated group than in control. In conclusion, rbST did not affect plasma concentrations of IGF-I and insulin, however, it did improve post-thaw sperm membrane integrity.

Neither bovine somatotropin nor growth hormone-releasing factor alters expression of thyroid hormone receptors in liver and mammary tissues

Physiological effects of thyroid hormones are mediated primarily by binding of triiodothyronine to specific nuclear receptors. Organ-specific changes in production of triiodothyronine from its prohormone, thyroxine, have been hypothesized to target the action of thyroid hormones on the mammary gland and play a role in mediating or augmenting a galactopoietic response to bovine somatotropin (bST). Additionally, tissue responsiveness to thyroid hormones may be altered by changes in the number or affinity of nuclear receptors for thyroid hormones. In the present study, effects of bST and bovine growth hormone-releasing factor (bGRF) on thyroid hormone receptors in liver and mammary gland were studied. Lactating Holstein cows received continuous infusions of bST or bGRF for 63 d or served as uninfused controls. Nuclei were isolated from harvested mammary and liver tissues and incubated with [(125)I]-triiodothyronine. Treatments did not alter the capacity or affinity of specific binding sites for triiodothyronine in liver or mammary nuclei. Evaluation of transcript abundance for thyroid hormone receptors showed that isoforms of thyroid hormone receptor or retinoid receptor (which may influence thyroid receptor action) expressed in the mammary gland were not altered by bST or bGRF treatment. Data do not support the hypothesis that administration of bST or bGRF alters sensitivity of mammary tissue by changing expression of thyroid hormone receptors.

Effect of early luteolysis in progesterone-based Al protocols in Bos indicus, Bos indicus x Bos taurus and Bos taurus heifers

The objective of this study was to evaluate the effects of treatment with an intravaginal progesterone-releasing device (CIDR) and estradiol benzoate (EB) on follicular dynamics in Bos indicus (n = 23), Bos taurus (n = 25), and cross-bred (n = 23) heifers. To assess the influence of reduced serum progesterone concentrations during 8 days of treatment with a progesterone-releasing device on follicular dynamics, half of the heifers received PGF at CIDR insertion (Day 0; 3 x 2 factorial design). Mean ( +/- S.E.M.) serum progesterone concentrations during CIDR treatment varied (P < 0.05) among genetic groups: B. indicus (5.4 +/- 0.1 ng/mL), B. taurus (3.3 +/- 0.0 ng/mL), and cross-bred (4.3 +/- 0.1 ng/mL). Maximum diameter of the dominant follicle (DF) was smaller (P < 0.01) in B. indicus heifers (9.5 +/- 0.5 mm) than in cross-bred (12.3 +/- 0.4 mm) or B. taurus heifers (11.6 +/- 0.5 mm). B. indicus experienced lower (P < 0.01) ovulation rate (39.1%) than did B. taurus (72.7%) and cross-bred (84.0%). Heifers treated with PGF on Day 0 had lower (P < 0.05) serum progesterone concentrations during progesterone treatment. The PGF treatment on Day 0 increased (P < 0.01) the diameter of the DF (11.9 +/- 0.4 mm vs. 10.5 +/- 0.4 mm). Moreover, greater (P = 0.02) ovulation rates (78.8 vs. 54.0%) occurred in heifers treated with PGF on Day 0. In summary, B. indicus heifers had greater serum progesterone concentrations, smaller DF diameter, and a lower ovulation rate compared to B. taurus heifers. Prostaglandin treatment on the day of CIDR insertion reduced serum progesterone during treatment, and resulted in increased maximum DF diameter and ovulation rate. (c) 2008 Elsevier Inc. All rights reserved.

Periodontal disease in adults with untreated congenital growth hormone deficiency: a case-control study

P>Aim The aim of this study was to investigate the possible associations between isolated growth hormone deficiency (IGHD) and periodontal attachment loss (PAL) in adults affected by congenital IGHD. Materials and methods Forty-five previously identified IGHD subjects were eligible for this study. The final study sample comprised 32 cases (gender:20M/12F; age:44.8 +/- 17.5) matched for age, gender, diabetes, smoking status and income to 32 controls (non-IGHD subjects). Participants were submitted to a full-mouth clinical examination of six sites per tooth and were interviewed using a structured, written questionnaire. Periodontitis was defined as proximal PAL >= 5 mm affecting >= 30% of teeth. Results No significant differences were observed in the percentage of sites with visible plaque between IGHD and non-IGHD subjects (59.4% versus 46.9%, p=0.32). IGHD subjects had significant less supragingival calculus (31.3% versus 59.4%, p=0.02) and more bleeding on probing (71.9% versus 18.8%, p < 0.01) than controls. PAL >= 5 mm was significantly more prevalent (100% versus 71.9%, p < 0.01) and affected more teeth (30.5% versus 6.7%, p < 0.01) in cases than in controls. After adjusting for supragingival calculus, IGHD cases had a higher likelihood of having periodontitis than controls (OR=17.4-17.8, 95% CI=2.3-134.9, p=0.004-0.005). Conclusion Congenital IGHD subjects have a greater chance of having PAL.